Benzyltriphenylphosphonium, salt with 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]bis[phenol] (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 October - 22 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in acordance with International guidelines and in accordance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Cas Number:

1478-61-1

Molecular formula:

C15H10F6O2

IUPAC Name:

4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley Crl:CD (SD) IGS BR strain rats were accepted on to the study

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: Yes- at study inititation
- Age at study initiation: ca. 9 weeks old
- Weight at study initiation: 301 - 375 g
- Fasting period before study: No
- Housing:
Initially, animals housed in groups of 4 or 5 in solid floor polypropylene cages (22 x 52 x 33 cm) with stainless steel mesh lids with softwood bedding.

During the mating phase, the non-recovery dose group animals were transferred to polypropylene grid floor cages (20 x 41.5 x 24 cm) suspended over trays lined with absorbent paper on a 1 male: 1 female basis within each dose group. Males were returned to original cages after successful mating.

Mated females were housed individually during gestation and lactation in solid floor polypropylene cages (20 x 41.5 x 24 cm) with stainless stell mesh lids and softwood flakes.

Recovery animals were housed in groups of 4 or 5 in solid florr polypropylene cages (22 x 52 x 33 cm) with stainless steel mesh lids and softwood flake bedding.

- Diet (e.g. ad libitum): free aces to pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories, Oxon, UK)
- Water (e.g. ad libitum): free acess to tap water
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: Diet certified (with CoA's). Neither diet or water considered to contain contaminants at a level that might have affected the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15 %
- Air changes (per hr): minimum of 15
- Photoperiod (hrs dark / hrs light): 12:12 light: dark

IN-LIFE DATES: From: 27 October 2009 To: 22 December 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Test material was prepared at the appropriate concentration as a suspension in Arachis oil BP. Stability and homogeneity of formulations was verified in a previous study. Fresh formulations were prepared every 2 weeks and stored at ca. +4 ºC in the dark. Subsamples were taken from each formualtion to verify concentration using a validated HPLC method. Measured concentrations were within ± 9 % of the nominal concentration throughout the study.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 7.5, 25 and 75 mg/mL prepared for 30, 100 and 300 mg/kg/day test groups
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): not reported
- Purity: not reported

Details on mating procedure:

Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots of each formulation were analysed using a validated HPLC method. Measured concentrations were within ± 9 % of the nominal concentration throughout the study.

Duration of treatment / exposure:

Test groups and controls: 55 consecutive days
Recovery groups: 42 consecutive days followed by a recovery phase of 14 days where no test item was administered.

Frequency of treatment:

Once, daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

High dose

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Recovery dose group

No. of animals per sex per dose:

Treatment groups: 12 males + 12 females per treatment group
Recovery groups: 5 males + 5 females per treatment group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose concentrations were based on the findings of a preliminary study conducted at 1000, 400 and 100 mg/kg bw.
- Rationale for animal assignment (if not random): not specified
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: yes, 14 days
- Section schedule rationale (if not random): not specified

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to 30 mins after dosing, one and 5 hours after dosing, during the working week. Animals were obsered immediately before dosing, soon after dosing and 1 hour after dosing, at weekends. During the treatment-free period, recovery animals were observed twice daily (once at weekends).
- Cage side observations checked in table ## were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: behavioural assessment: weekly
functional performance tests: prior to termination (in 5 males and 5 females per non-recovery treatment group)

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing then weekly (males until termination, females until mating was evident). Female bodyweights were then recorded on Day 0, 7, 14 and 20 post coitum, and on Day 0 and 4 post partum. During the mating phase, females were weighed daily untl mating was confirmed. Reovery animals were weighed before test initiation and weekly thereafter until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Non-recovery males: Day 14 and 42; recovery males: Day 56; non-recovery females: Day 14 and Day 4 post-partum; recovery females Day 56.
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Not specified
- How many animals: 5 per sex and test group
- Parameters checked in table ## were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as above
- Animals fasted: Not specified
- How many animals: 5 per sex and test group
- Parameters checked in table ## were examined.

URINALYSIS: Yes (males only)
- Time schedule for collection of urine: week 6
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table ## were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: not specified
- Dose groups that were examined: non-recovery animals (control, 30, 100 and 300 mg/kg/day)
- Battery of functions tested: grip strength, motor activity

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

OTHER:

Oestrous cyclicity (parental animals):

Group mean values for oestrous cycles for test and control group animals were determined. Smears were taken and evaluated daily.

Sperm parameters (parental animals):

Parameters examined in male parental (P0) generation:
testis weight, epididymis weight

Litter observations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 0 post partum. For each litter the following was recorded:

i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 0 and 4 post partum
iii) Sex of offspring on Days 0, 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring weights on Days 0 and 4 post partum

Postmortem examinations (parental animals):

Adult non-recovery males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult non-recovery females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. See Table 2 for measured parameters.

Postmortem examinations (offspring):

Surviving offspring (F1) were terminated via intracardiac overdose of sodium pentobarbitone. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum. See Table 2 for measured parameters.

Statistics:

The following parameters were subjected to statistical analysis

quantitative functional performance data
bodyweight and bodyweight change
food consumption
water consumption
haematology, blood chemistry and urinalysis
pre-coital intervals
gestation lengths
litter data- litter size, corpora lutea, implantation sites, litter weight
sex ratio
implantation losses, live birth index, viability indices, implantation index and delivery index
offspring bodyweight and bodyweight change
offspring surface righting
adult absolute and bodyweight relative organ weights

The following statistical procedures were used;

Data was assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparissons were conducted using Dunett's test. In case of recovery group data, the analysis used was a two-tailed t-test incorporating Levene's test for homogeneity of variance. Where Levene's test showed unequal variances the data was analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.

Non parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

Probability values (p) are presented as follows:
P<0.001***
P<0.01**
P<0.05*
P≥0.05 (not significant)

Histapathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes;

1. Chi-squared analysis for differences in the incidence of lesions occuring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Probability values (p) were calculated as follows:

P<0.001 +++ --- ***
P<0.01 ++ -- **
P<0.05 + - *
P<0.1 (+) (-) (*)
P≥0.1 (n.s.)

+/- difference vs. control

Reproductive indices:

The following parameters were calculated from the individual data during the mating period of the parental generation;

Pre-coital interval - calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility indices – mating index and fertility index calculated.
Gestation length - calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition index – gestation index calculated.

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

Implanatation losses – pre and post implanantation losses and impantation index.
Live birth and viability indices – live birth index, viability index and delivery index.
Sex ratio – sex ratio for surviving litter on Day 0, 1 and 4 post-partum and sex ratio at birth (total).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Dehydration and staining around the ano-genital region was evident for one female treted at 300 mg/kg/day on Days 6 and 7.
A second female showed dehydration on Day 7 and was hunched from Day 8-10.
A third female showed staining of the ano-genital region on Day 7.
Regresion of these signs was evident thereafter.

Other signs in the 300 mg/kg/day consisted of increased salivation after dosing and up to one hour after dosing on occasion of animals of either sex during the treatment period. Staining around the mouth were recorded and instances of noisy respiration noted in 5 males and 1 recovery female. Regression of these signs was evident followign cessation of treatment in recovery animals.

Increased salivation was observed in the 100 mg/kg/day group (both sexes) from week 3 with red/brown staining around the mouth observed. The incidence of signs was less in this group vs. the 300 mg/kg/day group. Increased salivation were detected up to 1 hour after dosing in the 30 mg/kg/day group (both sexes).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

300 mg/kg/day female displayed signs of hunched posture, lethargy, laboured and gasping breathing and tiptoe gait. Termination on Day 6 and resulting pathology of this individual concluded that the death was not material toxicity related but rather the result of an inappropriate dosing technique.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males;

300 mg/kg/day - significant reduction through the test period vs. controls. Bodyweight gains were statistically higher in the recovery group during the treatment free period. Reduced bodyweight increases throught the treatment period inevitably resulted in significantly lower mean bodyweights from Day 15 onwards.
100 mg/kg/day - significantly reduced bodyweight gain during the first 3 weeks of treatment.
30 mg/kd/day - no adverse effects noted.

Females;

300 mg/kg/day - 1 individual showed substancial weight loss (28 g) in week 1. Four other individuals showed slight bodyweight losses, resulting in a significant reduction in mean bodyweight gain vs. controls in week 1.
100 mg/kg/day - significantly lower bodyweight gains in the first week of treatment vs. controls. Bodyweight gain during gestation was comparable to controls. Mean bodyweights on Day 0 and 4 of lactation were significantly lower vs. controls.
30 mg/kg/day - significant reduction in bodyweight on Day 0 and 4 post partum.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males;

300 mg/kg/day - significant reduction in consumption during week 1 in non recovery (-22 %) and recovery (-28 %) animals vs. controls. Reduced consumption was also noted in both groups in week 2. Intake ws not measured during mating period however reduced intake was evident during this time. Reductions were evident in recovery animals through the remaining treatment period, although no difference was observed in the non-recovery group vs. controls. During the treatment-free period, treated animals intake was comparable to the controls.
100 mg/kg/day - significant reductions noted pre-mating when compared to controls. Intake improved and was comparable to controls after mating.
30 mg/kg/day - no adverse effects noted.

Females;

300 mg/kg/day - 25 % and 31 % reduction in intake during week 1 compared to controls in non-recovery and recovery groups. Recovery animals showed further reductions in intake up to week 5. Regression was evident during the treatment-free period.
100 mg/kg/day - 19 % reduction in week 1 and significant reductions through weeks 2 and 3 of the gestation period. Intake was comparable to controls during lactation.
30 mg/kg/day - Reduced intake during week 2 of gestation vs. controls.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males;

300 mg/kg/day - reduced efficieny vs control through weeks 1 - 7. Increased in efficiency (vs control) during treatment-free period
100 mg/kg/day - reduced efficiency vs control in week 1, although comparable to control though remainder of test
30 mg/kg/day - no difference vs controls

Females;

300 mg/kg/day - reduction in efficiency in week 1, comparable to controls through remainder of the test
100 mg/kg/day - reduction in week 1, comparable to controls through remainder of the test
30 mg/kg/day - reduction in week 1, comparable to controls through remainder of the test

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Males;

300 mg/kg/day - increased water intake noted in non-recovery individuals throught the whole test period versus control. Regression occuring in recovery males during treatment-free period.
100 mg/kg/day - increased water intake pre-mating (statistically) and post-mating (statistically, only in week 5) versus control.
30 mg/kd/day - increased water intake pre-mating (statistically) and post-mating (statistically, only in week 5) versus control.

Females

300 mg/kg/day - significant increase during week 1 and 2 in the recovery females (week 1-3, for non-recovery females) versus controls. Recovery evident during the treatment-free period.
100 mg/kg/day - significant increase during week 1versus controls. Increase during gestation and early lactation , although not statistically significant.
30 mg/kg/day - not significantly different vs controls.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males;

300 mg/kg/day - lower (not statistically significant) haemoglobin and erythrocyte values observed on Day 14. Significant (slight) reduction in reticulocyte counts versus controls on Day 14. At Day 42, significant reduction in haemoglobin and erythrocytes counts were oberved versus controls. Hematocrit counts also lower (although not significantly) at this timepoint. Regression of these findings was observed in the recovery males during the treatment-free period with the exception of erythrocyte counts. Significant increase in mean cell volume and mean cell haemoglobinwas observed in the recovery males versus controls.
30 and 100 mg/kg/day - No changes versus control.

Females;

300 mg/kg/day - no significant differences versus controls.
100 mg/kg/day - significant reduction in mean cell haemoglobin compared to controls on Day 14 (slight and in the absence of a dose-related response and other haemotological changes at this level, this finding was considered to have been incidental).
30 mg/kg/day - no significant change versus controls

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males;

300 mg/kg/day (pre-mating) - significant reduction in blood albumin on Day 14 (pre-mating). Lower A/G ratios and increased alanine aminotransferase levels were also evident, although not significantly so. Significant increase in blood urea levels versus controls. Significantly, blood cholesterol was reduced during pre-mating, versus control values.
300 mg/kg/day (pre-termination) - significant increase in blood urea levels versus controls with reduction in blood albumin levels evident also. Reduction of blood cholesterol and increase in alanine aminotrasferase continued at significant levels.

Regression of changes observed during the treatment-free period in recovery males with slight reduction in plasma bilirubin noted, versus controls. Slight increases in A/G ratio and plasma chloride levels noted.

100 mg/kg/day - significant reduction in blood albumin on Day 14 (pre-mating) and Day 42. Reduction in blood choloesterol on Day 42 versus controls with an increse in alanine aminotransferase also noted.
30 mg/kg/day - reduced blood cholesterol pre-mating

Females;

300 mg/kg/day (pre-mating) - significant reduction in blood albumin and A/G ratios on Day 14 (pre-mating). Significant increase in alanine aminotransferase levels and reduction in plasma chloride levels compared to controls observed. Significantly, blood cholesterol was reduced during pre-mating, versus control values although a dose response curve was not apparent.
300 mg/kg/day (pre-termination) - Day 4 post-partum blood levels were not significantly changed versus the controls.
30 and 100 mg/kg/day - reduced blood cholesterol pre-mating (significnat)

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant changes versus controls observed in any of the treated males

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Behavioural assessment - Noisy respiration noted in one female of 300 mg/kg/day group- also noted in clinical observations.

Functional observations - no significant changes in treated animals versus controls.

Functional performance - no significant changes in treated animals versus controls.

Sensory reactivity assessment - no significant changes in treated animals versus controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mammary gland

Tubuloalveolar differentiation of mamary tissue was seen in males treated at all three concentrations, although only statistically significant data was observed at 300 mg/kg/day. There was no evidence of regression of the observation in the recovery males after the treatment-free period. Minimal glandular hyperplasia of the mammary tissue was seen for four non-pregnant females treated at 300 mg/kg/day. This may have been an effect of treatment. Hyperplasia was not seen in recovery control or 300 mg/kg/day females following completion of the treatment free period.

Ovaries

Follicular cysts were seen among non-pregnant females in the 300 mg/kg/day group, this may have been an effect in the absence of directly comparable controls. Follicular cysts were seen in the recovery females versus controls suggesting that the effect had not regressed.

Testes

Leydig cell atrophy was seen in relation to treatment for males treated with 300 mg/kg/day and at 100 mg/kg/day, although not statistically significant at this level. The condition regressed among the recovery males after the treatment free period. Moderate or severe testicular atrophy was seen for two recovery males. This condition does occur spontaneously among laboratory maintained rats and there was no evidence to suggest this was a treatment related consequence.

Seminal vesicles/ coagulating gland

Reduced secretory content as indicated by smaller organ size was seen in relation to treatment for males treated with 300 mg/kg/day and 100 mg/kg/day compared to control, but not statistically significant at 30 mg/kg/day. There was no evidence of regression of the condition among 300 mg/kg/day males after the treatment-free period has elapsed.

Prostate

Reduced secretory content as indicated by smaller organ size was seen in relation to treatment for males treated with 300 mg/kg/day and 100 mg/kg/day, but not at 30 mg/kg/day. There was no convincing regression observed in the recovery males.

Liver

Centrilobular hepatocyte enlargement was seen in relation to treatment for males treated with 300 and 100 mg/kg/day with the effects also evident at 30 mg/kg/day (statistically significant). Females were also affected at 300 mg/kg/day at (not statistically significant) and 100 mg/kg/day (statistically significant). Regression was observed in both sexes in the recovery animals.

Kidneys

A greater incidence of higher grades of severity of groups of basophillic tubules and tubular dilatation were seen as a consequence of treatment for males with 300 mg/kg/day compared to controls (statistically significant)) but not at other dose levels. Not observed (convincingly) for females. Both conditions regressed in the recovery group.

Adrenal glands

Cortical vacuolation is relatively common in lab-maintained rats and is especially prevalent among males and more rarely seen among females. The condition was significantly less prevalent among males treated at 300 mg/kg/day and 100 mg/kg/day. Although this may be a spurious group distribution of incidence and severity grades and effect of treatment on the adrenal cortex cannot be excluded. A similar effect was not seen in the females. A group differential was maintained among recovery animals suggesting that any effec was not fully regressed.

Lungs

Groups of alveolar macrophages were prevalent among control animals of either sex and grades of severity ranged from minimal to moderate. Such a macrophage response was rather greater than might normally be seen in te control animals of this age. The incidence and severity of alveolar macrophage populations wa significantly lower for males and femlaes treated at 300 mg/kg/day (stat. analysis not performed on females) and 100 mg/kg/day. Although such incidnece and severity could be fortuitous, an effect of treatment cannot be excluded. No evidence of alveolar macrophage accumulation after the treatment-free period had elapsed, suggesting regression of any effect.

Pituitary

Vacuolation of pars anterior cells is commonly seen among male rats but it is rarely present in female rats of this age. The prevalence and severity grades of vacuolation were normal or slightly above normal for control males but significantly lower for males treated at 300 mg/kg/day, indicating a dose-related effect. This effect was not seen in females or males treated at other concentrations. There was no evidence of regression in the recovery males.

Uterus/ cervix

Dilation of the uterine horn, with or without keratinisation in the cervix was found in one female treated with 30 mg/kg/day and one female treated with 100 mg/kg/day which displayed in utero total litter losse and one non-pregnant female treatde with 30 mg/kg/day, 2 non-pregnant females treated with 100 mg/kg/day and 2 nonpregnant females from the 300 mg/kg/day dose groups. This simply represents normal cyclical changes in the female rat. In addition, necrotic contenets were present in the uterus from one female treated at 100 mg/kg/day. This female showedd a corpus luteum and implantation site during the post mortum procedure, therefore this was considered to represent resorption of the foetuses.

Vagina

Hyperplasia of the vaginal epithelium was sen for 4 non-pregnant females at 300 mg/kg/day, but allowing for cyclical changes, there was insufficient evidence to suggest an effect of treatment. Similarly, higher grades of severity of vascuolar degeneration of the post partum vaginal epithelium as normal conversion from mucinous to non-mucinous morpholgy were seen among intermediate dose females compared to controls. There was no convincing effect of the treatment in this study. Keratinisation of the vaginal epithelium is a normal cyclical change in the female rat.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See above

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

One female treated with 300 mg/kg/day showed a continuous anestrus interval and also failed to mate. Another female treated with 300 mg/kg/day showed extended oestrus. This female mated but did not achieve pregnancy. These events were considered unusual.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were no pregnant females observed at 300 mg/kg/day. Seven pregnant females were evident at 100 mg/kg/day and three females at this dose level which showed positive evidence of mating but did not achieve pregnancy. One female treated with 100 mg/kg/day showed evidence of mating and post-mortem examinations revealed the presence of a corpus luteum and an implantation site, however, this female did not produce a live litter. All females treated with 30 mg/kg/day showed positive evidence of mating, although two females did not achieve pregnancy. One female treated at this dose level did not deliver a live litter but showed two dead foetuses in utero during the post-mortem procedure. Pregnancy was achieved in the eleven control females which showed positive evidence of mating.

Details on results (P0)

Reproduction;

For male animals, a ‘No Observed Adverse Effect Level’ (NOAEL) could not be established because of infertility at 300 mg/kg/day and reduced fertility at 100 and 30 mg/kg/day.

For female animals treated with 300 mg/kg/day, pregnancy was not achieved. A NOAEL was not achieved for reproductive toxicity because of reduced pregnancy rates for parental females treated with 100 and 30 mg/kg/day compared to parental females from the control group.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

for P0 parental

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable

Remarks:

reduced fertility observed in both male and female groups at the lowest tested concentration.

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Daily clinical observations of offspring did not reveal any clinical signs considered to be related to test material toxicity. The clinical signs observed were those commonly observed in offspring in reproductive studies of this type, and were not considered to represent adverse effects of treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

For interim death offspring, macroscopic findings were confined to autolytic changes, with the exception of three offspring from one 30 mg/kg/day litter, which were cannibalised. These findings are commonly observed in interim death offspring in reproductive studies of this type, and were considered not to represent an effect of treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A slight but statistically significant reduction in Day 4 post partum litter weights was evident for 30 mg/kg/day litters when compared to controls (P<0.05). The significance achieved was minimal and in the absence of a dose-related response, this isolated intergroup difference was considered to have arisen incidentally and was of no toxicological importance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Surface righting assessments on Day 1 post partum did not reveal any significant intergroup differences between litters from treated animals when compared to litters from controls.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

yes

Any other information on results incl. tables

Table 4:      Average body weights and body weight gains during 56 days of treatment (P0)

Dose rate (mg/kg/day)	Male Body Weights at Day (g)									Total Weight Gain (g)
	1	8	15	22	29	36	43	50	57
Male
Control	340	370	390	412	436	456	461	-	-	120
Low	342	370	393	411	437	455	466	-	-	124
Mid	340	359	375	395	421	439	449	-	-	108
High	343	356	371	384	404	418	425	-	-	83*
Recovery control	346	387	422	455	484	507	528	548	556	210
Recovery high	344	352	367*	382*	395*	406*	412*	445*	463*	120**
Female Body Weights (Weight Gain) During (g);
Dose rate (mg/kg/day)	Maturation (at day)			Gestation (at day)				Lactation (at day)		Total Weight Gain (g)
	1	8	15	0	7	14	20	0	4
Control	241	250 (9)	256 (6)	271	304 (33)	340 (36)	421 (81)	324	332 (8)	-
Low	238	241 (3)	246 (5)	253	284 (31)	314 (30)	377* (63)	301*	300** (-1)	-
Mid	233	233 (1**)	239 (6*)	248	282 (34)	312 (31)	382 (70)	292*	301* (9)	-
High	242	240 (-2**)	250 (9)	-	-	-	-	-	-	-
Recovery control	236	245	254	262	271	280	283	290	295	59
Recovery high	236	235	242	249	257	263	267	279	279	43

*  Significantly different (p <0.05) from the control.

** Significantly different (p <0.01) from the control.

*** Significantly different (p <0.001) from the control.

Table 5:      Selected haematology, clinical chemistry and pathology findings (P0)

Doses (mg/kg/day)	Control	30	100	300	Control	30	100	300
	male				female
Number of animals/group	5	5	5	5	5	5	5	5 (day 56 recovery)
Haematology(day 42 for males and day 4 post-partum for females)
- Hb (g/dl)	16.6	16.2	16.1	15.2**	13.3	12.7	12.4	15.5
- RBC (10^12/L)	8.89	8.78	8.46	8.09**	6.91	6.62	6.53	8.29
- Hct (%)	47.5	46.9	46.9	44.9	39.6	37.4	36.9	45.4
- MCH (pg)	18.7	18.5	19.0	18.7	19.4	19.1	19.0	18.7
- MCV (fl)	53	53	55	55	57	56	57	55
- MCHC (g/df)	34.9	34.7	34.2	33.8	33.7	33.8	33.6	34.1
- WBC (10^9/L)	10.8	9.2	11.3	8.0	9.0	7.5	12.1	7.3
Blood chemistry(day 42 and day 4 post-partum for females)
- Urea (mg/dl)	29	32	29	37**	35	40	50	44
- Glucose (mg/dl)	147	159	140	153	118	122	128	163
- Tot. Prot. (g/dl)	6.57	6.45	6.34	6.42	5.65	5.74	5.57	7.58
- Albumin (g/dl)	3.6	3.5	3.3*	3.3**	3.2	3.2	3.1	4.2
- A/G ratio	1.21	1.17	1.14	1.09	1.34	1.30	1.20	1.26
- Na+ (mmol/L)	150	150	150	150	151	149	152	152
- K+ (mmol/L)	4.58	4.32	4.46	4.16	4.80	4.08	4.59	4.20
- Cl- (mmol/L)	104	103	105	104	106	105	104	107
Pathology	male				female
Number of animals/group	12	12	12	12	12	12	12	11^a
- External,mass under right forelimb	0	0	1	0	0	0	0	0
- Internal,epididymides: small	0	0	1	0	0	0	0	0
- Internal,right kidney: hydronephrosis	0	0	1	0	0	0	0	0
- Internal,lungs: mottled appearance	0	0	1	0	0	0	0	0
- Internal,mandibular lymph nodes: enlarged	0	0	1	0	0	0	0	0
- Internal,mass: approx.. 2 cm, spherical, containing green/yellow substance	0	0	1	0	0	0	0	0
- Internal,seminal vesicles: small	0	0	0	4	-	-	-	-
- Internal,prostate: small	0	0	0	4	-	-	-	-
- Internal,testes: small	0	0	1	0	-	-	-	-
- Internal,adrenals: pale	0	0	0	0	0	1	0	0
- Internal,cervical lymph nodes: enlarged	0	0	0	0	0	0	0	1
- Internal,lungs: reddened	0	0	0	0	1	0	0	1
- Internal,mass: approx. 1.5 cm, containing white coloured viscous liquid	0	0	0	0	0	0	0	1
- Internal,ovaries: dark red discolouration	-	-	-	-	0	0	0	1
- Internal,stomach: sloughing- glandular region	0	0	0	0	0	1	0	0
- Internal,Uterus: 1 dead foetus in each horn	-	-	-	-	0	1	0	0
- No abnormalities	12	12	9	8	11	11	12	9

Statistics: Anova + Dunnetts tests (two sided): * P,0.05   ** P,0.01

^aone female killed in extremis on Day 6 exhibited the following pathological signs; adrenals (dark and enlarged), gastro-intestinal tract (gaseous distention), liver (dark), lungs (reddened), stomach (sloughing- non-glandular region), thoracic cavity (containing liquid), mass under left forelimb (containing foods and liquid).

Table 6:      Absolute and relative organ weights (P0)

		Males (non-recovery)				Females (non-recovery)			Females (recovery)
DAILY DOSE (mg/kg bw/day)		Control	30	100	300	Control	30	100	Control	300
NUMBER OF ANIMALS		12	12	12	12	7-11	7-11	7-11	5	4-5
BODY WEIGHT (g)^a		461	466	449	425	332	301	304	295	279
BRAIN
Absolute Weight^a	g	2.0179	2.0543	2.0266	2.0070	1.9028	1.8502	1.9078	1.9121	1.8922
Per Body Weight^a	%	0.4423	0.4419	0.4535	0.4754	0.5755	0.6164*	0.6282*	0.6530	0.6810
ADRENALS
Absolute Weight^a	g	0.0562	0.0562	0.0527	0.0636	0.0760	0.0705	0.0700	0.0628	0.0571
Per Body Weight^a	%	0.0122	0.0121	0.0118	0.0150**	0.0229	0.0234	0.0229	0.0214	0.0202
EPIDIDYMIDES
Absolute Weight^a	g	1.2917	1.3495	1.2100	1.0385***	n.a.^b	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Body Weight^a	%	0.2824	0.2900	0.2695	0.2448**	n.a.^b	n.a.^b	n.a.^b	n.a.^b	n.a.^b
HEART
Absolute Weight^a	g	1.7237	1.6602	1.5566	1.5537	1.3490	1.1218*	1.1404*	1.0731	1.1116
Per Body Weight^a	%	0.3738	0.3563	0.3477	0.3666	0.4053	0.3726	0.3762	0.3661	0.3994
KIDNEYS
Absolute Weight^a	g	3.1949	3.2058	3.0922	3.1681	1.9178	1.8878	1.9243	1.9781	1.8940
Per Body Weight^a	%	0.6952	0.6877	0.6910	0.7431	0.5778	0.6261	0.6316	0.6713	0.6802
LIVER
Absolute Weight^a	g	15.6764	15.5205	14.9837	15.8695	12.7095	11.6779	11.9166	9.3369	9.3088
Per Body Weight^a	%	3.3934	3.3273	3.3404	3.7250*	3.8193	3.8884	3.9040	3.1752	3.3435
SPLEEN
Absolute Weight^a	g	0.7653	0.7845	0.7309	0.6956	0.6289	0.5669	0.5916	0.5168	0.4972
Per Body Weight^a	%	0.1659	0.1688	0.1634	0.1638	0.1888	0.1890	0.1941	0.1746	0.1784
TESTES
Absolute Weight^a	g	3.4920	3.5732	3.2223	3.1171*	n.a.^b	n.a.^b	n.a.^b	n.a.^b	n.a.^b
Per Body Weight^a	%	0.7641	0.7701	0.7177	0.7350	n.a.^b	n.a.^b	n.a.^b	n.a.^b	n.a.^b
THYROID
Absolute Weight^a	g	0.0178	0.0189	0.0196	0.0170	0.0149	0.0118	0.0135	0.0141	0.0127
Per Body Weight^a	%	0.0039	0.0041	0.0043	0.0040	0.0045	0.0040	0.0044	0.0049	0.0046
THYMUS
Absolute Weight^a	g	0.3756	0.3925	0.3732	0.3393	0.2620	0.2118	0.2171	0.3301	0.2999
Per Body Weight^a	%	0.0823	0.0838	0.0840	0.0801	0.0790	0.0706	0.0715	0.1118	0.1072
OVARIES
Absolute Weight^a	g	n.a.^b	n.a.^b	n.a.^b	n.a.^b	0.2076	0.0935	0.0958	0.0851	0.0712
Per Body Weight^a	%	n.a.^b	n.a.^b	n.a.^b	n.a.^b	0.0594	0.0312	0.0313	0.0289	0.0256
UTERUS
Absolute Weight^a		n.a.^b	n.a.^b	n.a.^b	n.a.^b	0.8779	0.7427	0.8500	0.9308	0.6539
Per Body Weight^a		n.a.^b	n.a.^b	n.a.^b	n.a.^b	0.2642	0.2484	0.2787	0.3140	0.2356

^aGroup means at the end of terminal necropsy are shown.

Table 7:       Mating Performance, Fertility and Gestation Length (P0)

Dose Level (mg/kg/day)	Number of males paired	Number of females			Pre-coital interval (Days)
		Paired	Mated	Pregnant	1	2	3	4	5	6	14
0 (control)	12	12	11	11	1	2	4	3	0	0	1
30	12	12	12	10	5	1	3	3	0	0	0
100	12	12	11	8	4	1	3	1	1	0	0
300	12	11	10	0	3	3	0	0	1	2	1

Dose level (mg/kg/day)	Mating index (%)	Fertility index (%)	Gestation Length (Days)						Females with live offspring	Gestation Index (%)
			22	22.5	23	23.5	24	Not confirmed
0 (control)	91.7	100	2	2	3	4	0	0	11	100
30	100.0	83.3	1	2	3	2	1	0	9	90
100	91.7	63.6	0	1	5	0	0	1	7	87.5
300	90.9	0	-						-	-

Table 8:       Litter and Offspring Bodyweight Data

Dose group (mg/kg/day)		Number of corpora lutea	Number of implantation sites	Total number of offspring born	Number of live offspring		Litter weight (g)		Offspring weight (g)				Offspring bodyweight change (g)
					Day 0	Day 4	Day 0	Day 4	Day 0		Day 4		Days 0 - 4
									♂	♀	♂	♀	♂	♀
0 (control)	mean	16.7	14.1	13.1	13.0	12.8	85.5	118.1	6.9	6.5	9.8	9.2	2.9	2.7
	sd	3.6	1.9	2.5	2.6	2.7	11.4	12.8	0.8	0.8	1.5	1.7	0.8	1.0
	n	11	10	11	11	11	11	11	11	11	11	11	11	11
30	mean	14.7	12.1	10.9	10.2	10.0	68.6	97.7*	7.1	6.6	10.5	9.6	3.4	3.0
	sd	5.8	3.8	3.3	2.6	2.5	11.2	12.4	0.8	0.7	1.8	1.4	1.0	0.8
	n	9	7	9	9	9	9	9	9	9	9	9	9	9
100	mean	14.0	10.4	11.7	11.3	10.9	76.7	100.6	7.2	6.6	10.0	9.3	2.8	2.7
	sd	7.8	4.8	3.4	3.7	3.5	23.3	22.4	0.9	0.7	1.8	1.5	1.0	1.1
	n	7	7	7	7	7	7	7	7	7	7	7	7	7
300	mean
	sd
	n

 

Table 9:       Implantation Losses and Offspring Survival Indices

Dose Group (mg/kg/day)		Pre-Implantation Loss (%)	Post-Implantation Loss (%)	Live Birth (%)	Viability Index (%)
0 (control)	mean	11.9	9.5	99.1	98.4
	sd	13.2	10.3	3.0	3.7
	n	10	10	11	11
30	mean	10.6	7.8	95.8	98.0
	sd	18.2	7.6	12.5	4.1
	n	7	7	9	9
100	mean	18.7	22.9	95.6	96.3
	sd	16.8	35.5	8.5	6.4
	n	7	7	7	7
300	mean
	sd
	n

Table 10:       Sex Ratio - Group Mean Litter Values

Dose Group (mg/kg/day)		Sex Ratio (% Males) on (Post Partum) Day;
		At Birth	1	4
0 (control)	mean	47.5	47.9	48.8
	sd	9.0	8.7	9.4
	n	11	11	11
30	mean	45.4	45.2	44.7
	sd	14.9	14.7	14.7
	n	9	9	9
100	mean	48.7	48.0	47.9
	sd	10.1	10.9	10.9
	n	7	7	7
300	mean	-	-	-
	sd
	n

Dose Group (mg/kg/day)	Number of Litters	Sex Ratio (Fraction of Males) on (Post Partum) Day;
		At Birth	1	4
0 (control)	11	0.48	0.48	0.49
30	10	0.44	0.43	0.43
100	12	0.48	0.46	0.46
300	0	-	-	-

Table 11:       Offspring Clinical Observations- Summary Indices

Dose Level (mg/kg/day)	Number of Litters	Clinical Observation	Number of Offspring (Number of Litters) Affected (Post Partum) Day;
			0	1	2	3	4	5
0 (control)	11	Abdominal bruising	0	0	0	1F(1)	0	0
		Bruised dorsal surface	1M(1)	1M(1)	0	0	0	0
		Found dead	1F(1)	0	0	0	0	2F(2)
		No milk present in stomach	1F(1)	1F(1)	1F(1)	2F(2)	3F(3)	0
		Missing	0	0	1F(1)	0	1F(1)	1M(1)
		Missing tail	1F(1)	1F(1)	1F(1)	1F(1)	1F(1)	1F(1)
		Pale	1F(1)	1F(1)	1F(1)	1F(1)	1F(1)	0
		Physical injury to ventral surface	1F(1)	1F(1)	1F(1)	1F(1)	0	0
		Scab formation on ventral surface	1F(1)	1F(1)	1F(1)	1F(1)	0	0
		Small	1F(1)	1F(1)	1F(1)	1F(1)	3F(3)	0
		Swollen left hindlimb	0	0	1M(1)	0	0	0
		Swollen right forelimb	0	0	1M(1)	1M(1)	0	0
		No abnormalities	(6)	(7)	(5)	(6)	(7)	(8)
30	9	Bruised snout	1F(1)	0	0	0	0	0
		Cannibalised	3(1)	0	0	0	0	0
		Found dead	1F, 2M(1)	0	0	0	0	0
		No milk present in stomach	0	0	0	1F(1)	0	0
		Missing	0	0	1M(1)	1F(1)	0	0
		Pale	0	1M(1)	0	0	0	0
		Small	1F(1)	1F(1)	1F(1)	1F(1)	1F(1)	1F(1)
		No abnormalities	(6)	(7)	(7)	(7)	(8)	(8)
100	7	Bruised snout	1F(1)	0	0	0	0	0
		Cold	1F, 2M(1)	0	0	0	1F(1)	0
		Found dead	1F, 2M(2)	0	0	0	0	1F(1)
		Missing	0	1M(1)	0	0	2M(1)	1F(1)
		No milk present in stomach	1F(1)	0	1F(1)	1F(1)	1F(1)	0
		Pale	1M(1)	1M(1)	1M(1)	0	0	0
		Small	2F(2)	2F(2)	2F(2)	1F(1)	9F, 7M(3)	8F, 7M (2)
		Swollen right hindlimb	0	0	1F(1)	1F(1)	1F(1)	0
		Weak	0	0	0	0	1F(1)	0
		No abnormalities detected	(4)	(5)	(4)	(4)	(3)	(4)
300	0	-	-	-	-	-	-	-

Table 12:       Reflexological Responses for Offspring - Group Mean Litter Values

Dose level (mg/kg/day)		Surface Righting Reflex (% passed)
0 (control)	mean	91.2
	sd	7.4
	n	11
30	mean	90.9
	sd	11.4
	n	9
100	mean	90.3
	sd	13.3
	n	7
300	mean	-
	sd	-
	n	-

Table 13:       Necropsy Findings for Offspring - Group Indices

Observation	Number of Offspring (Litters) Affected at Dose Level (mg/kg/day)
	0 (control)	30	100	300
INTERIM DEATHS
Number of offspring	3F(3)	2M, 1F, 3U(1)	2M, 2F(3)	-
Autolytic changes detected	2F(2)	2M, 1F(1)	2M, 2F(3)	-
Cannibalised	0	3U(1)	0	-
No abnormalities detected	1F(1)	0	0	-
TERMINAL KILL
Missing tail	1F(1)	0	0	-
Scab formation on ventral surface	1F(1)	0	0	-
Small	0	1F(1)	2F(2)	-
LITTER WITH NO ABNORMALITIES	9	8	5	-

Applicant's summary and conclusion

Conclusions:

For male animals, a ‘No Observed Adverse Effect Level’ (NOAEL) could not be established because of the microscopic changes in the mammary gland at all treatment levels, infertility at 300 mg/kg/day and reduced fertility at 100 and 30 mg/kg/day.

For female animals treated with 300 mg/kg/day, pregnancy was not achieved. For parental females, a NOAEL for systemic toxicity was achieved at 30 mg/kg/day because effects at 30 mg/kg/day were not considered to represent an adverse health effect for systemic toxicity. A NOAEL was not achieved for reproductive toxicity because of reduced pregnancy rates for parental females treated with 100 and 30 mg/kg/day compared to parental females from the control group.

No adverse effects were evident in the offspring from the 100 and 30 mg/kg/day dose groups compared to the controls. Litter sizes and litter weights for treated animals were compared to controls and no treatment related effects on development were observed at these dose levels. For F1 offspring, the ‘No Observed Effect Level’ (NOEL) and NOAEL was 100 mg/kg/day under these test conditions.

Executive summary:

In a 1-generation reproduction study (OECD 422) bisphenol-AF (99.9 % purity) was administered daily to 58 male and 58 female Sprague-Dawley rats (Crl:CD (SD) IGS BR strain) in Arachis oil BP by oral gavage at dose levels of 30, 100 and 300 mg/kg bw/day for 55 days. An additional group of recovery animals were treated according to the dose groups, previously described, up to the point of sacrifice, at which time the treatment was discontinued. After fourteen days without treatment, the recovery males and females were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.  The recovery group allowed investigators to establish if any of the adverse effects observed in the treatment groups were recoverable after cessation of treatment.

For P0 male animals, a ‘No Observed Adverse Effect Level’ (NOAEL) could not be established because of the microscopic changes in the mammary gland at all treatment levels which did not regress following cessation of treatment.  Similarly, for toxicity to reproduction, infertility at 300 mg/kg/day and reduced fertility at 100 and 30 mg/kg/day meant that a NOAEL could not be established for the male rat.

For P0 female animals treated with 300 mg/kg/day, pregnancy was not achieved. For parental females, a NOAEL for systemic toxicity was achieved at 30 mg/kg/day because effects at 30 mg/kg/day were not considered to represent an adverse health effect for systemic toxicity.  A NOAEL was not achieved for reproductive toxicity because of reduced pregnancy rates for parental females treated with 100 and 30 mg/kg/day compared to parental females from the control group.

No adverse effects were evident in the offspring from the 100 and 30 mg/kg/day dose groups compared to the controls. Litter sizes and litter weights for treated animals were compared to controls and no treatment related effects on development were observed at these dose levels. For F1 offspring, the ‘No Observed Effect Level’ (NOEL) and NOAEL was 100 mg/kg/day under these test conditions.

Although the mechanism of test material toxicity may not be identified from this study alone, it is suspected that the test material is oestrogenic, based on the findings in the reproductive organs for both male and female rats. To summarise, significant effects on fertility were evident at 300 and 100 mg/kg/day, resulting in no pregnancies at 300 mg/kg/day and fewer pregnancies at 100 mg/kg/day and at 30 mg/kg/day.

This study is acceptable and satisfies the guideline requirement for a 1-generation reproductive study OECD 422 in rats.